Figure 1: Cell pellet from 500 ml E. coli BL21(DE3) with EhPTP expressed after induction and spin down steps
The cell pellet obtained had a mass of 3.07g, which may or may not correlate to the amount of EhPTP protein obtained. The result of this expression will not become apparent until analyzation through purification and characterization. The pellet is an indication of the total amount of protein in the culture, including cell walls, organelles, and other miscellaneous solids (such as contaminants) in the solution. MIDI Prep for PmCherry:
Figure 2 & 3: Absorbance spectra at 280nm for protein from midiprep results of PmCherry in elution buffer. From nanodrop. 3 Trials are shown in table below the spectra. Concentrations shown in ng/microliter
The midiprep protocol was preformed twice, once for each pellet. The amount of plasmid obtained had an average of 34.01 and 49.1 ng/ul for sample 1 and 2, respectively. Both samples were sent to be sequenced to confirm their identities.
Agarose Gel:
Figure 4& 5: Agarose gel with (in order of columns), a 100 bp ladder, Kaan's samples A-D, another 100bp ladder, and Tiger's samples A-D. Samples A-D contained 0.3, 3, 30, and 0 ng of the purple protein coding sequence , respectively. 10 ul of a 4:5 dilution of samples in each well. Figure 4 (black) was not color inverted but no DNA was visible. Figure 5 is color inverted.
Two Gels were ran, and in the second trial, DNA was visible! The problem with the first gel was faulty or not enough ethidium bromide. The darkest band in figure 5 is near the 700 bp range, which is the length of the amplified gene. Samples A-C show a similar darkness of bands, but sample c has more smudging indicating contamination. Interestingly, sample D shows a band, likely from some contamination from sample C when loading the wells. There should have been a decrease in color from A-D as less DNA was in each well, but this was not apparent. It is likely that the PGBR22 gene was successfully amplified through PCR.
RE digests:
Two digests were performed, one of PGBR22 and one of pmCherry. The first digest of PGBR22 yielded no results except for the DNA ladder. It is thought that the uncut plasmid was contaminated with nuclease. PmCherry, which was shown to be visible on a gel uncut, was then digested with EcoRI, EagI, BsaI, and all 3.
Analysis: NEB cutter confirms only the "all 3" lane. Banding for lanes 2, 3, and 4 should have been the same with a 5 kb linear fragment. There may have been a mutation that caused EcoRI to cut twice, and uncut may show 2 bands because some plasmid supercoiled and some did not. Lane 4 is odd; there is one prominent fragment that is shorter than the linear 5kb fragment.
MIDI Prep for PmCherry:
Agarose Gel:
Two Gels were ran, and in the second trial, DNA was visible! The problem with the first gel was faulty or not enough ethidium bromide. The darkest band in figure 5 is near the 700 bp range, which is the length of the amplified gene. Samples A-C show a similar darkness of bands, but sample c has more smudging indicating contamination. Interestingly, sample D shows a band, likely from some contamination from sample C when loading the wells. There should have been a decrease in color from A-D as less DNA was in each well, but this was not apparent. It is likely that the PGBR22 gene was successfully amplified through PCR.
RE digests:
Two digests were performed, one of PGBR22 and one of pmCherry. The first digest of PGBR22 yielded no results except for the DNA ladder. It is thought that the uncut plasmid was contaminated with nuclease. PmCherry, which was shown to be visible on a gel uncut, was then digested with EcoRI, EagI, BsaI, and all 3.
Analysis: NEB cutter confirms only the "all 3" lane. Banding for lanes 2, 3, and 4 should have been the same with a 5 kb linear fragment. There may have been a mutation that caused EcoRI to cut twice, and uncut may show 2 bands because some plasmid supercoiled and some did not. Lane 4 is odd; there is one prominent fragment that is shorter than the linear 5kb fragment.